Journal of Nanobiotechnology · 6 authors, 3 centres
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This study identifies that plasma exosome miR-26b-3p is significantly upregulated in children with idiopathic short stature (ISS) and impairs longitudinal bone growth by suppressing chondrocyte proliferation and endochondral ossification. The mechanism involves miR-26b-3p targeting the AKAP2/ERK1/2 signaling axis, and miR-26b-3p shows diagnostic potential with an AUC of 0.823. These findings provide a new molecular basis for ISS pathogenesis and suggest a potential therapeutic target.
**Background:** Idiopathic short stature (ISS) is defined as height below the mean by more than 2 standard deviations or below the 3rd percentile, after excluding known causes. ISS accounts for 30–80% of short stature cases, yet its etiology remains unclear. Known genetic mutations (GHR, SHOX, FGFR3, ACAN, NPR2) explain only approximately 5% of cases. Recombinant human growth hormone therapy shows variable efficacy and potential safety concerns, including increased incidence of bone/cartilage tumors and cerebrovascular events. Circulating exosomes are key mediators of intercellular communication, but their role in ISS pathogenesis had not been reported.
**Methods:** Plasma exosomes were isolated from 70 ISS children and 70 healthy controls (mean age 8.92±0.34 vs. 8.39±0.28 years; mean height 120.2±1.72 vs. 132.1±1.57 cm). Exosomes were characterized by transmission electron microscopy, Nano-Sight analysis, and western blotting for CD63 and CD9. Human chondrocytes were co-cultured with ISS or control exosomes. High-throughput microRNA sequencing was performed on 5 pairs of samples to identify differentially expressed miRNAs. RT-qPCR, western blotting, CCK-8 assays, flow cytometry, ALP activity assays, luciferase reporter assays, in situ hybridization, and immunohistochemistry were used. In vivo experiments used tail vein injection of miR-26b-3p-overexpressing exosomes or adenoviral vectors in Sprague-Dawley rats, with BrdU and calcein labeling to assess growth plate proliferation and bone growth.
**Key Results:** ISS plasma exosomes suppressed normal human chondrocyte proliferation (CCK-8), caused G0/G1 cell cycle arrest, and downregulated hypertrophic differentiation markers (COL10A, RUNX2) and osteogenic genes (OCN, OPN), with reduced ALP activity. miRNA sequencing identified 13 differentially expressed miRNAs (6 upregulated, 7 downregulated; fold change ≥2.0, P<0.05). miR-26b-3p showed the highest upregulation and was confirmed by RT-qPCR. ROC analysis yielded an AUC of 0.823 (95% CI 0.754–0.892); at a cutoff >1.173, specificity was 71.4% and sensitivity was 85.7%. Silencing miR-26b-3p in ISS exosomes reversed the inhibition of chondrocyte proliferation and endochondral ossification. Overexpression of miR-26b-3p reproduced the inhibitory effects. Luciferase assays confirmed AKAP2 as a direct target of miR-26b-3p. AKAP2 overexpression rescued the miR-26b-3p-induced suppression. miR-26b-3p overexpression did not alter total ERK1/2 or ER-α mRNA/protein levels but significantly reduced p-ERK1/2 and p-ER-α levels. In vivo, miR-26b-3p-overexpressing exosomes localized to growth plate chondrocytes, downregulated AKAP2, reduced COL10A, RUNX2, OCN, and OPN expression, decreased growth plate height, reduced BrdU-positive proliferating cells, and significantly lowered femur, tibia, and overall body length in rats.
**Clinical Implications:** This study is the first to demonstrate that plasma exosome miR-26b-3p is upregulated in ISS and impairs longitudinal bone growth via the AKAP2/ERK1/2 axis. miR-26b-3p shows promise as a diagnostic biomarker for ISS (AUC 0.823). The AKAP2/ERK1/2 pathway represents a potential therapeutic target for ISS, offering an alternative to recombinant growth hormone therapy. Limitations include the inability to obtain growth plate chondrocytes from ISS children due to medical ethics constraints.